Search

New variants of SARS-CoV-2 are continuously being reported. To curtail the spread of this virus, it is essential to find an efficient and potent vaccine. Here, we report in silico designing of a protein (ferritin: FR) nanocage fused with multiple epitopes identified using the immuno-informatics approach and high-throughput screening. Employing computational approaches, we identified potential epitopes from membrane, nucleocapsid, and envelope proteins of SARS-CoV-2 and docked them on the selected human leukocyte antigen Class I and II receptors, then the stability of the complexes was assessed using molecular dynamics simulation studies. We have engineered chimeric ferritin nanocage, chm66FR, with the nested peptide of 10 epitopes by replacing the loop region at the 66(th) position of the nanocage, then its stability was confirmed using metadynamics simulation. Further, we used the homotrimeric '6-helical bundle' of the spike protein to engineer the chimeric 6HB (chm6HB). The chm6HB is, engineered with three epitope peptides, mounted on the N-terminal trimeric interface of the chm66FR to generate the chm6HB-chm66FR, which contains 15 epitope peptides. Chimeric FR nanocages and the chm6HB could be potential vaccine candidates against strains of SARS-CoV-2. These multivalent and multiple epitopes protein nanocages and scaffolds could mount both humoral and T-cell mediated immune responses against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

New variants of SARS-CoV-2 are continuously being reported. To curtail the spread of this virus, it is essential to find an efficient and potent vaccine. Here, we report in silico designing of a protein (ferritin: FR) nanocage fused with multiple epitopes identified using the immuno-informatics approach and high-throughput screening. Employing computational approaches, we identified potential epitopes from membrane, nucleocapsid, and envelope proteins of SARS-CoV-2 and docked them on the selected human leukocyte antigen Class I and II receptors, then the stability of the complexes was assessed using molecular dynamics simulation studies. We have engineered chimeric ferritin nanocage, chm66FR, with the nested peptide of 10 epitopes by replacing the loop region at the 66(th) position of the nanocage, then its stability was confirmed using metadynamics simulation. Further, we used the homotrimeric '6-helical bundle' of the spike protein to engineer the chimeric 6HB (chm6HB). The chm6HB is, engineered with three epitope peptides, mounted on the N-terminal trimeric interface of the chm66FR to generate the chm6HB-chm66FR, which contains 15 epitope peptides. Chimeric FR nanocages and the chm6HB could be potential vaccine candidates against strains of SARS-CoV-2. These multivalent and multiple epitopes protein nanocages and scaffolds could mount both humoral and T-cell mediated immune responses against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

New variants of SARS-CoV-2 are continuously being reported. To curtail the spread of this virus, it is essential to find an efficient and potent vaccine. Here, we report in silico designing of a protein (ferritin: FR) nanocage fused with multiple epitopes identified using the immuno-informatics approach and high-throughput screening. Employing computational approaches, we identified potential epitopes from membrane, nucleocapsid, and envelope proteins of SARS-CoV-2 and docked them on the selected human leukocyte antigen Class I and II receptors, then the stability of the complexes was assessed using molecular dynamics simulation studies. We have engineered chimeric ferritin nanocage, chm66FR, with the nested peptide of 10 epitopes by replacing the loop region at the 66(th) position of the nanocage, then its stability was confirmed using metadynamics simulation. Further, we used the homotrimeric '6-helical bundle' of the spike protein to engineer the chimeric 6HB (chm6HB). The chm6HB is, engineered with three epitope peptides, mounted on the N-terminal trimeric interface of the chm66FR to generate the chm6HB-chm66FR, which contains 15 epitope peptides. Chimeric FR nanocages and the chm6HB could be potential vaccine candidates against strains of SARS-CoV-2. These multivalent and multiple epitopes protein nanocages and scaffolds could mount both humoral and T-cell mediated immune responses against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Researchers from IIT Hyderabad have found a new molecule which can potentially treat Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig's disease.

Researchers from IIT Hyderabad have found a new molecule which can potentially treat Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig's disease.

Researchers from IIT Hyderabad have found a new molecule which can potentially treat Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig's disease.

Researchers from IIT Hyderabad have found a new molecule which can potentially treat Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig's disease.

Researchers from IIT Hyderabad have found a new molecule which can potentially treat Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig's disease.

Kinases are involved in important biological pathways, cell signaling and cell cycle control. Many kinases in pathogenic organisms are responsible for the organism survival and causing infection. Calcium dependent protein kinase (CDPK) is one such kinase present exclusively in apicomplexan parasites. In respond to the calcium signals, these CDPKs get activated and regulate protein secretion, activation of motility, cell invasion and egress. Since humans don’t have CDPKs homolog and are critical for survival of the pathogen, CDPKs acts as a good drug target in diseased conditions. Traditionally available inhibitors are reversible inhibitors; these reversible inhibitors are less potent. On the other hand, covalent inhibitor binds irreversible and form targeted covalent bond with the amino acid residue in the binding pocket. CDPKs don’t have covalent inhibitor. We tried to generate specific reversible and irreversible (covalent) inhibitors against CDPKs by in-silico structure analysis. Using Schrödinger, maestro software derivatives are generated, docked and molecular dynamic simulation studies carried out. The designed covalent inhibitor may be more potent and will have more resident time compare to reversible inhibitors available in the market. Covalent inhibitors may bind stably to the protein and may prevent activation of CDPK even in the presence of calcium signal.

Kinases are involved in important biological pathways, cell signaling and cell cycle control. Many kinases in pathogenic organisms are responsible for the organism survival and causing infection. Calcium dependent protein kinase (CDPK) is one such kinase present exclusively in apicomplexan parasites. In respond to the calcium signals, these CDPKs get activated and regulate protein secretion, activation of motility, cell invasion and egress. Since humans don’t have CDPKs homolog and are critical for survival of the pathogen, CDPKs acts as a good drug target in diseased conditions. Traditionally available inhibitors are reversible inhibitors; these reversible inhibitors are less potent. On the other hand, covalent inhibitor binds irreversible and form targeted covalent bond with the amino acid residue in the binding pocket. CDPKs don’t have covalent inhibitor. We tried to generate specific reversible and irreversible (covalent) inhibitors against CDPKs by in-silico structure analysis. Using Schrödinger, maestro software derivatives are generated, docked and molecular dynamic simulation studies carried out. The designed covalent inhibitor may be more potent and will have more resident time compare to reversible inhibitors available in the market. Covalent inhibitors may bind stably to the protein and may prevent activation of CDPK even in the presence of calcium signal.

In all the living systems, reactive oxygen species (ROS) metabolism provides resistance against internal and external oxidative stresses. Auranofin (AF), an FDA-approved gold [Au(I)]-conjugated drug, is known to selectively target thiol-reductases, key enzymes involved in ROS metabolism. AF has been successfully tested for its inhibitory activity through biochemical studies, both in vitro and in vivo, against a diverse range of pathogens including protozoa, nematodes, bacteria, and so forth. Cocrystal structures of thiol-reductases complexed with AF revealed that Au(I) was coordinately linked to catalytic cysteines, but the mechanism of transfer of Au(I) from AF to catalytic cysteines still remains unknown. In this study, we have employed computational approaches to understand the interaction of AF with thiol-reductases of selected human pathogens. A similar network of interactions of AF was observed in all the studied enzymes. Also, we have shown that tailor-made analogues of AF can be designed against selective thiol-reductases for targeted inhibition. Molecular dynamics studies show that the AF-intermediates, tetraacetylthioglucose (TAG)-gold, and triethylphosphine (TP)-gold, coordinately linked to one of catalytic cysteines, remain stable in the binding pocket of thiol-reductases for Leishmania infantum and Plasmodium falciparum (PfTrxR). This suggests that the TP and TAG moieties of AF may be sequentially eliminated during the transfer of Au(I) to catalytic cysteines of the receptor.

In all the living systems, reactive oxygen species (ROS) metabolism provides resistance against internal and external oxidative stresses. Auranofin (AF), an FDA-approved gold [Au(I)]-conjugated drug, is known to selectively target thiol-reductases, key enzymes involved in ROS metabolism. AF has been successfully tested for its inhibitory activity through biochemical studies, both in vitro and in vivo, against a diverse range of pathogens including protozoa, nematodes, bacteria, and so forth. Cocrystal structures of thiol-reductases complexed with AF revealed that Au(I) was coordinately linked to catalytic cysteines, but the mechanism of transfer of Au(I) from AF to catalytic cysteines still remains unknown. In this study, we have employed computational approaches to understand the interaction of AF with thiol-reductases of selected human pathogens. A similar network of interactions of AF was observed in all the studied enzymes. Also, we have shown that tailor-made analogues of AF can be designed against selective thiol-reductases for targeted inhibition. Molecular dynamics studies show that the AF-intermediates, tetraacetylthioglucose (TAG)-gold, and triethylphosphine (TP)-gold, coordinately linked to one of catalytic cysteines, remain stable in the binding pocket of thiol-reductases for Leishmania infantum and Plasmodium falciparum (PfTrxR). This suggests that the TP and TAG moieties of AF may be sequentially eliminated during the transfer of Au(I) to catalytic cysteines of the receptor.

In all the living systems, reactive oxygen species (ROS) metabolism provides resistance against internal and external oxidative stresses. Auranofin (AF), an FDA-approved gold [Au(I)]-conjugated drug, is known to selectively target thiol-reductases, key enzymes involved in ROS metabolism. AF has been successfully tested for its inhibitory activity through biochemical studies, both in vitro and in vivo, against a diverse range of pathogens including protozoa, nematodes, bacteria, and so forth. Cocrystal structures of thiol-reductases complexed with AF revealed that Au(I) was coordinately linked to catalytic cysteines, but the mechanism of transfer of Au(I) from AF to catalytic cysteines still remains unknown. In this study, we have employed computational approaches to understand the interaction of AF with thiol-reductases of selected human pathogens. A similar network of interactions of AF was observed in all the studied enzymes. Also, we have shown that tailor-made analogues of AF can be designed against selective thiol-reductases for targeted inhibition. Molecular dynamics studies show that the AF-intermediates, tetraacetylthioglucose (TAG)-gold, and triethylphosphine (TP)-gold, coordinately linked to one of catalytic cysteines, remain stable in the binding pocket of thiol-reductases for Leishmania infantum and Plasmodium falciparum (PfTrxR). This suggests that the TP and TAG moieties of AF may be sequentially eliminated during the transfer of Au(I) to catalytic cysteines of the receptor.

UHRF1 is a multi-domain protein comprising of a tandem tudor domain (UHRF1 TTD), a PHD finger, and a SET and RING-associated (UHRF1 SRA) domain. It is involved in the maintenance of CG methylation, heterochromatin formation and DNA repair processes. Isothermal titration calorimetry binding studies of UHRF1 TTD with unmodified and methylated lysine histone peptides establishes that the UHRF1 TTD binds dimethylated lysine 9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that, together, UHRF1’s TTD and PHD (UHRF1 TTD-PHD) preferentially recognizes dimethyllysine status. Importantly, it was found that in the binding pocket of UHRF1, the Asp145 which determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. In contrast, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of H3K9me2 by the UHRF1 TTD-PHD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 (H3R2K9me2) by the UHRF1 TTD-PHD. Electrophoretic mobility shift assay and fluorescence polarization binding studies indicate that the UHRF1 SRA binds to all the oxidation products of 5-methyl cytosine (5mC) but exhibits lower affinity towards 5-carboxyl cytosine (5caC) and 5-formyl cytosine (5fC). Subtle variations of key residues at the binding pocket could determine status specific recognition of histone methyllysine by the reader domains. Thus, further studies are required to unravel the possibility of combinatorial recognition of H3R2K9me2, and 5hmCG DNA by the UHRF1 and role of combinatorial recognition on UHRF1 functions especially for CG methylation maintenance and heterochromatin formation.

UHRF1 is a multi-domain protein comprising of a tandem tudor (UHRF1 TTD), a PHD finger, and a SET and RING-associated domain. It is required for the maintenance of CG methylation, heterochromatin formation and DNA repair. Isothermal titration calorimetry binding studies of unmodified and methylated lysine histone peptides establish that the UHRF1 TTD binds dimethylated Lys9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that TTD-PHD of UHRF1 (UHRF1 TTD-PHD) preferentially recognizes dimethyl-lysine status. Importantly, we show that Asp145 in the binding pocket determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. Interestingly, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of K9me2 by the UHRF1 TTD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 by the UHRF1 TTD-PHD. We propose that subtle variations of key residues at the binding pocket determine status specific recognition of histone methyl-lysines by the reader domains.

UHRF1 is a multi-domain protein comprising of a tandem tudor domain (UHRF1 TTD), a PHD finger, and a SET and RING-associated (UHRF1 SRA) domain. It is involved in the maintenance of CG methylation, heterochromatin formation and DNA repair processes. Isothermal titration calorimetry binding studies of UHRF1 TTD with unmodified and methylated lysine histone peptides establishes that the UHRF1 TTD binds dimethylated lysine 9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that, together, UHRF1’s TTD and PHD (UHRF1 TTD-PHD) preferentially recognizes dimethyllysine status. Importantly, it was found that in the binding pocket of UHRF1, the Asp145 which determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. In contrast, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of H3K9me2 by the UHRF1 TTD-PHD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 (H3R2K9me2) by the UHRF1 TTD-PHD. Electrophoretic mobility shift assay and fluorescence polarization binding studies indicate that the UHRF1 SRA binds to all the oxidation products of 5-methyl cytosine (5mC) but exhibits lower affinity towards 5-carboxyl cytosine (5caC) and 5-formyl cytosine (5fC). Subtle variations of key residues at the binding pocket could determine status specific recognition of histone methyllysine by the reader domains. Thus, further studies are required to unravel the possibility of combinatorial recognition of H3R2K9me2, and 5hmCG DNA by the UHRF1 and role of combinatorial recognition on UHRF1 functions especially for CG methylation maintenance and heterochromatin formation.

UHRF1 is a multi-domain protein comprising of a tandem tudor (UHRF1 TTD), a PHD finger, and a SET and RING-associated domain. It is required for the maintenance of CG methylation, heterochromatin formation and DNA repair. Isothermal titration calorimetry binding studies of unmodified and methylated lysine histone peptides establish that the UHRF1 TTD binds dimethylated Lys9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that TTD-PHD of UHRF1 (UHRF1 TTD-PHD) preferentially recognizes dimethyl-lysine status. Importantly, we show that Asp145 in the binding pocket determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. Interestingly, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of K9me2 by the UHRF1 TTD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 by the UHRF1 TTD-PHD. We propose that subtle variations of key residues at the binding pocket determine status specific recognition of histone methyl-lysines by the reader domains.

UHRF1 is a multi-domain protein comprising of a tandem tudor (UHRF1 TTD), a PHD finger, and a SET and RING-associated domain. It is required for the maintenance of CG methylation, heterochromatin formation and DNA repair. Isothermal titration calorimetry binding studies of unmodified and methylated lysine histone peptides establish that the UHRF1 TTD binds dimethylated Lys9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that TTD-PHD of UHRF1 (UHRF1 TTD-PHD) preferentially recognizes dimethyl-lysine status. Importantly, we show that Asp145 in the binding pocket determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. Interestingly, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of K9me2 by the UHRF1 TTD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 by the UHRF1 TTD-PHD. We propose that subtle variations of key residues at the binding pocket determine status specific recognition of histone methyl-lysines by the reader domains.

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analysed ATX, SUVH, SUVR, ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity towards histone 3 lysine 4 (H3K4) and might catalyse the tri-methylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse mono- or di-methylation of H3K9. Except for SDG728, which may tri-methylate H3K9, all SUVH paralogues in rice may catalyse mono- or di-methylation. ZmSET11, ZmSET31, SDG713, SDG715 and SDG726 proteins are predicted to be catalytically inactive because of an incomplete SAM binding pocket and a post-SET domain. E(Z) homologues can tri-methylate H3K27 substrate, which is similar to the EZH2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein-protein interaction and PRC2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre- and post-SET domains in HKMTs of the aforementioned plant species lead to variations in class-specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity. This article is protected by copyright. All rights reserved.

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analysed ATX, SUVH, SUVR, ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity towards histone 3 lysine 4 (H3K4) and might catalyse the tri-methylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse mono- or di-methylation of H3K9. Except for SDG728, which may tri-methylate H3K9, all SUVH paralogues in rice may catalyse mono- or di-methylation. ZmSET11, ZmSET31, SDG713, SDG715 and SDG726 proteins are predicted to be catalytically inactive because of an incomplete SAM binding pocket and a post-SET domain. E(Z) homologues can tri-methylate H3K27 substrate, which is similar to the EZH2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein-protein interaction and PRC2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre- and post-SET domains in HKMTs of the aforementioned plant species lead to variations in class-specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity. This article is protected by copyright. All rights reserved.

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

Evasion of innate immune recognition is one of the key strategies for persistence of Helicobacter pylori, by virtue of its ability to modulate or escape the host innate immune receptors and signaling pathways. C-type lectin receptors (CLRs) predominantly expressed by macrophages are pivotal in tailoring immune response against pathogens. The recognition of glyco or carbohydrate moieties by Mincle (Macrophage inducible C-type lectin) is emerging as a crucial element in anti-fungal and anti-mycobacterial immunity. Herein, we demonstrate the role of Mincle in modulation of innate immune response against H. pylori infection. Our results revealed an upregulated expression of Mincle which was independent of direct host cell contact. Upon computational modelling, Mincle was observed to interact with the Lewis antigens of H. pylori LPS and possibly activating an anti-inflammatory cytokine production, thereby maintaining a balance between pro- and anti-inflammatory cytokine production. Furthermore, siRNA mediated knockdown of Mincle in human macrophages resulted in up regulation of pro-inflammatory cytokines and consequent down regulation of anti-inflammatory cytokines. Collectively, our study demonstrates a novel mechanism employed by H. pylori to escape clearance by exploiting functional plasticity of Mincle to strike a balance between pro-and anti-inflammatory responses ensuring its persistence in the host.

Evasion of innate immune recognition is one of the key strategies for persistence of Helicobacter pylori, by virtue of its ability to modulate or escape the host innate immune receptors and signaling pathways. C-type lectin receptors (CLRs) predominantly expressed by macrophages are pivotal in tailoring immune response against pathogens. The recognition of glyco or carbohydrate moieties by Mincle (Macrophage inducible C-type lectin) is emerging as a crucial element in anti-fungal and anti-mycobacterial immunity. Herein, we demonstrate the role of Mincle in modulation of innate immune response against H. pylori infection. Our results revealed an upregulated expression of Mincle which was independent of direct host cell contact. Upon computational modelling, Mincle was observed to interact with the Lewis antigens of H. pylori LPS and possibly activating an anti-inflammatory cytokine production, thereby maintaining a balance between pro- and anti-inflammatory cytokine production. Furthermore, siRNA mediated knockdown of Mincle in human macrophages resulted in up regulation of pro-inflammatory cytokines and consequent down regulation of anti-inflammatory cytokines. Collectively, our study demonstrates a novel mechanism employed by H. pylori to escape clearance by exploiting functional plasticity of Mincle to strike a balance between pro-and anti-inflammatory responses ensuring its persistence in the host.

Evasion of innate immune recognition is one of the key strategies for persistence of Helicobacter pylori, by virtue of its ability to modulate or escape the host innate immune receptors and signaling pathways. C-type lectin receptors (CLRs) predominantly expressed by macrophages are pivotal in tailoring immune response against pathogens. The recognition of glyco or carbohydrate moieties by Mincle (Macrophage inducible C-type lectin) is emerging as a crucial element in anti-fungal and anti-mycobacterial immunity. Herein, we demonstrate the role of Mincle in modulation of innate immune response against H. pylori infection. Our results revealed an upregulated expression of Mincle which was independent of direct host cell contact. Upon computational modelling, Mincle was observed to interact with the Lewis antigens of H. pylori LPS and possibly activating an anti-inflammatory cytokine production, thereby maintaining a balance between pro- and anti-inflammatory cytokine production. Furthermore, siRNA mediated knockdown of Mincle in human macrophages resulted in up regulation of pro-inflammatory cytokines and consequent down regulation of anti-inflammatory cytokines. Collectively, our study demonstrates a novel mechanism employed by H. pylori to escape clearance by exploiting functional plasticity of Mincle to strike a balance between pro-and anti-inflammatory responses ensuring its persistence in the host.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.